Silencing of Nicotiana benthamiana Xrn4p exoribonuclease promotes tombusvirus RNA accumulation and recombination

The cytosolic 5′-to-3′ exoribonuclease Xrn1p plays a major role in recombination and degradation of Tomato bushy stunt tombusvirus (TBSV) replicon (rep)RNA in yeast, a model host (Serviene, E., Shapka, N., Cheng, C.P., Panavas, T., Phuangrat, B., Baker, J., and Nagy, P.D., 2005. Genome-wide screen identifies host genes affecting viral RNA recombination. Proc. Natl. Acad. Sci. U. S. A. 102(30), 10545–10550.). To test if the plant cytosolic 5′-to-3′ exoribonuclease Xrn4p, similar to the yeast Xrn1p, could also affect TBSV recombination, in this paper, we silenced XRN4 in Nicotiana benthamiana, an experimental host. The accumulation of tombusvirus genomic RNA and repRNA increased by 50% and 220%, respectively, in XRN4-silenced N. benthamiana. We also observed up to 125-fold increase in the emergence of new recombinants and partly degraded viral RNAs in the silenced plants. Using a cell-free assay based on a yeast extract, which supports authentic replication and recombination of TBSV, we demonstrate that the purified recombinant Xrn1p efficiently inhibited the accumulation of recombinants and partly degraded viral RNAs. Altogether, the data from a plant host and cell-free system confirm a central role for the plant cytosolic 5′-to-3′ exoribonuclease in TBSV replication, recombination and viral RNA degradation.